Effect of Stress-Free Therapy on immune system: Induction of Interleukin 10 expression in lymphocytes through activation of CD19⁺ CD24^hi CD38^hi regulatory B Cells

Abstract

Background and aims: Mild thermal treatment with “Pinpoint Plantar Long-wavelength Infrared Light Irradiation (PP-LILI)” named as Stress-Free Therapy^® increases peripheral-deep body temperature and blood flow, and improves multiple disorders including hyperpiesia, type II diabetes and cardiovascular patients. Immunomodulatory effects of PP-LILI were investigated.

Materials and methods: Seven healthy individuals and 4 people with underlying medical condition (UMC) participated in this study. Participants were given PP-LILI stimuli twice a week over 3 weeks and followed with placebo stimuli over 3 weeks. This set of sessions was repeated 3 times. For analyses, fresh peripheral mononuclear cells from participants were stained with fluorescencedye conjugated monoclonal antibodies and changes in populational compositions and IL-10 expression levels were observed by flow cytometry.

Results: Distinct expression of IL-10 in lymphocytes was induced by PP-LILI from the second session in the healthy individuals. This induction was terminated during the following placebo sessions. PP-LILI induced activation of CD19⁺ CD24^hi CD38^hi regulatory B cells in every session prior to induce the IL-10 in major lymphocytes. Activated regulatory B cells in the individuals with UMC decreased as same levels of healthy individuals after second PP-LILI session and re-activated with the stimuli. Significant population changes in neither regulatory T cells nor proinflammatory IL-17A expressing CD4⁺ T cells were observed.

Conclusions: PP-LILI is a potent immunomodulatory inducer that activates regulatory B cells and consequent IL-10 expression in lymphocytes. Moreover, its stimulatory intervals down-regulate the higher activation of regulatory B cells and lymphocyte's IL-10 expression occurred by UMC to the healthy people's level.

Introduction

We have previously reported significant clinical effects of a mild thermal treatment with “Pinpoint Plantar Long-wavelength Infrared Light Irradiation (PP-LILI)” named as Stress-Free Therapy^{® 1–3)}. Although this treatment is non-invasive, it increases peripheral-deep body temperature and blood flow, and improves multiple disorders including hyperpiesia, type II diabetes and cardiovascular patients without medications. Those effects may be consequences of integrated changes in homeostasis-modulating factors in blood stream since significant reductions of cortisol and adrenocorticotropic hormone have been observed.

PP-LILI gives pin-point surface stimuli, and thus it is similar to moxibustion and acupuncture despite PP-LILI is not invasive nor hurt the skin. Moxibustion and acupuncture are the traditionally developed invasive therapy in China for curative and analgia ^{4, 5)}. Its significance has been appreciated and spread worldwide as an alternative treatment and complementary therapeutic interventions for pain and diseases. In the last decades, attributive effects of those stimuli have been shed light on, and it was partly revealed that those stimuli affect our immune system through modulation of pro-inflammatory cytokines and inflammatory suppressing cytokine IL-10 ^6–8).

Interleukin-10 calms immune system and consequently suppresses inflammatory symptoms including swelling, redness, hyperthermia and pain ⁹⁾, and thus IL-10 may be a primary mediator of moxibustion and acupuncture in pain treatment and anti-inflammatory therapy. In previous reports, Han et al showed moxibustion increases serum IL-10 level while it decreases pro-inflammatory cytokine IL-8 and improves symptoms of ulcerative colitis in rat model ⁶⁾. Zhu et al showed that electroacupuncture treatment sustained inflammation-induced serum IL-10 level while proinflammatory cytokine IL-6 level was decreased during conservative therapy for the acute pancreatitis patients ⁸⁾. In contrast, Joos et al observed decreases of serum IL-10 and IL-6 levels while an increase of IL-8 level along with improvement of symptoms in allergic asthma patients treated with acupuncture ¹⁰⁾. So, the contribution of IL-10 in the effect of pin-point stimuli remains elusive. Those inconsistencies in observations may have resulted from differences in experimental settings, diseases and location of body points stimulated. However, at least the contribution of IL-10 as a mediator for suppressing inflammations should be indispensable because its distinct function has been confirmed by the experimental immunology ⁹⁾.

In this paper, we describe the repeated PP-LILI stimuli induces a distinct IL-10 expression in lymphocytes of healthy individuals. This induction was terminated in the following non-stimulatory sessions and the levels of IL-10 expression decreased to basal levels in both healthy individuals and the people with UMC. In addition, PP-LILI stimuli induce activation of CD19⁺ CD24^hi CD38^hi regulatory B cells (B regs) just after the second stimulus of every stimulatory session including the first sequence of sessions, and the duration of activation was extended along with the iteration of stimulatory sessions. Moreover, high background levels of activated B reg cells seen in the people with UMC were decreased to the same levels of healthy people after second and third sessions of PP-LILI stimuli. Meanwhile the regulatory T cell subpopulations did not show significant response to the stimuli. Since B regs play primary roles for suppressing inflammation and autoimmune disorders through activation of immunomodulatory cells ¹¹⁾, PP-LILI stimuli may not only induce the competence of lymphocytes for IL-10 expression, but it may improve or reset the systemic immune activity through the B reg activation.

Materials and Methods

Volunteers

Seven healthy volunteers (4 males and 3 females; age 35 – 69, mean age 48.3 ± 11.3) and 4 individuals with UMC (4 males; age 35 – 76, mean age 58.8 ± 20.1, one renal failure, two hypertensive and one atopic syndrome) were enrolled. No daily habit including diet, drinking alcohol and Japanese style hot-tub bathing activity were restricted.

Treatments and peripheral blood sampling

The volunteers were subjected to blood sampling, and 6 to 10 ml of peripheral blood was withdrawn through cubital vein on the first day (day 0) and subsequently 11 times (day 7, 21, 35, 49, 56, 69, 77, 91, 98, 112 and 118) before stimulatory treatment. PP-LILI was delivered to the volunteers resting in spine position for 15 minutes with the irradiation probes placed at four points of body surface. Two probes were placed on the respective soles at the intersections of the lines; one drawn along the center between the first and second metatarsal bones and another drawn vertically from the top of medial malleolus. Remaining probes were placed on acupuncture points; one resides on Zusanli point (World Health Organization (WHO) established number: ST36) that is at the outside of left knee, and another on Zhongwan point (WHO established number: CV12) that is at the center between lower end of the sternum and umbilicus. Irradiation was controlled at less perceptible by Stress-Free device^® (Controlled Medical Device certificate no. 224AFBZX00075000; probe diameter 2 mm; inflared wavelength 9,000 to 12,000 nm; output 30 mW). A session for treatments with PP-LILI stimuli were given two times per week over three weeks (typically started on Thursday and ended on Monday) followed by three weeks of session for placebo stimuli where the probes were placed as in PP-LILI treatment without irradiation (Figure 1A).

Figure 1.

Experimental designs for stimulatory sequences and measurements of targeted populations from PBMC.

(A) Volunteers were treated over 118 days with three sequences of stimulatory sessions. Every sequence comprised a session of PP-LILI stimuli over three weeks (SF session; solid thin vertical arrows show respective stimulus given; 6 times of stimuli in the session) and a session of placebo stimuli (Placebo session; dotted thin vertical arrows show respective stimulus given; 6 or 8 times of quasi-stimuli in the session). Peripheral blood samples were withdrawn before stimulus on the day indicated with thick vertical arrows. Dotted thick arrows show blood-withdrawal to check results of placebo stimuli and no-stimulation. Solid thick arrows show blood-withdrawal to check results of PP-LILI stimuli.

(B) Gating strategy for identification of target subpopulations from lymphocytes. Peripheral blood mononuclear cells (PBMCs) were prepared from individual volunteers and were stained with monoclonal antibody mixtures against following cellular antigens; 1^st mixture contains anti-CD19, CD24, CD38 and intracellular IL-10, 2^nd mixture contains anti-CD4, CD25 and intracellular FoxP3, 3^rd mixture contains anti-CD8, CD45RA, CD56, CD161 and FoxP3, 4^th mixture contains anti-CD4 and intracellular IL-17A. The PBMCs were then subjected for flow cytometry as described in Materials and Methods. Interleukin 10 expressing levels in lymphocytes were calculated on the basis of fluorescence intensity (sequence a). CD19⁺ CD24^hi CD38^hi progenitor regulatory B cells (pro-B regs) and its activated IL-10 expressing subpopulation (activated B regs), CD4⁺ CD25^hi FoxP3⁺ regulatory T cells (CD4⁺ T regs), CD8⁺ CD161⁻ CD56⁺ CD45RA⁺ FoxP3⁺ regulatory T cells (CD8⁺ T regs), and IL-17A expressing CD4⁺ T cells (CD4⁺ IL-17A⁺ T cells) were similarly identified from lymphocytes (sequences b, c, d, e, respectively).

Preparation of peripheral blood mononuclear cells and staining

Peripheral blood mononuclear cells (PBMC) were prepared from freshly withdrawn heparinized peripheral blood by using HetaSep™ reagent (STEMCELL technologies Inc., Vancouver, Canada) as described in manufacturer's instruction. Briefly, five part of peripheral blood was mixed well with one part of HetaSep™ reagent and centrifuged at 90 × g for 20 minutes (min) without decelerator. Then, yellowish upper layer was collected and diluted with phosphate buffered saline (PBS) for four times and centrifuged at 120 × g for 5 min. PBMC were collected as pellet and washed with PBS for two times and suspended in 1.5 ml of PBS containing 2% heat-inactivated fetal calf serum (FCS) and 0.05% sodium azide. Aliquots of 100 µl of PBMC suspensions were dispensed in wells of 96-well polystyrene plates and reacted with antibody mixture for cell surface antigens for 30 min at room temperature, respectively. Cells were then centrifuged and washed with 200 µl of PBS, fixed with PBS containing 2% paraformaldehyde for 10 min, pierced with PBS containing paraformaldehyde and 0.5% Triton X-100 for 10 min, washed with PBS twice and activated residues were blocked in PBS containing 2% FCS for 30 min. Subsequently the cells were washed, stained with secondary antibody mixture containing anti-intracellular proteins for 30 min, washed and submitted to flow cytometry to detect aimed cell populations.

Antibody reagents and flow cytometry

Anti-CD19 (clone HIB19, murine IgG1), anti-CD4 (clone RPA-T4, murine IgG1), and anti-CD8a (clone RPA-TA, murine IgG1) antibodies conjugated with phycoerythrin (PE) -Cy7, anti-CD45RA (clone HI100, murine IgG2b) conjugated with fluorescein isothiocyanate (FITC), anti-CD25 (clone BC96, murine IgG1) and anti-CD161 (clone HP-3G10, murine IgG1) conjugated with PE, and all isotype control antibodies were purchased from TONBO biosciences (San Diego, USA). Anti-CD24 (clone ML5, murine IgG2a) conjugated with FITC, anti-CD38 (clone HIT2, murine IgG1) conjugated with PerCP-Cy5, anti-CD56 (clone HCD56, murine IgG1) conjugated with PE-Cy7, and anti-IL-10 (clone JES3-9D7, rattus IgG1) and anti-FoxP3 (clone 150D, murine IgG1) conjugated with allophycocyanin (APC) were purchased from BioLegend Inc. (San Diego, USA). All antibody reagents were adequately mixed and diluted in PBS containing 2% FCS and 0.05% sodium azide for 100 times. Equivalent amount of diluent was mixed with cell suspension for staining. For flow cytometry, JSAN fluorescence activated cell sorter (FACS; Bay Bioscience Co. Ltd., Kobe, Japan) was employed and following parameters were measured without compensation; forward light scatter (FSC) for particle size, side scatter (SSC) for internal complexity of particle, Fluorescence (FL)-1A for FITC, FL-2A for PE, FL-3A for PerCP-Cy5.5, FL-4A for PE-Cy7, FL-5A for APC and FL-6A for APC-Cy7. Obtained raw data were analyzed lymphocyte subpopulation with AppSan ver.3.1 for Windows 7 and FlowJo ver. X 10.0.7 and ver. X 10.0.8 for Mac OSX. To analyze IL-10 expressing cells, five sequences of gating strategies were applied on the lymphocytes (Figure 1B). That were; common gate for lymphocyte population with FSC and SSC; (a) mean fluorescence intensities of IL-10 (FL-5) in whole lymphocyte population, (b) CD19⁺ population was gated on CD19 (FL-4) and CD24 (FL-1), and then CD24^hi CD38^hi population was separated from CD19⁺ population on FL-1 and CD38 (FL-3), and then its IL-10 expressing population was measured on FL-5 versus cell count histogram; (c) CD4⁺ CD25^hi subpopulation was gated out from lymphocyte population by FL-6 and FL-2, and its FoxP3 positive subpopulation was calculated in FL-5 histogram; (d) CD8⁺ CD161⁻ subpopulation was gated out from lymphocyte by FL-2 and FL-6, then CD45RA⁺ CD56⁺ subpopulation was gated out and FoxP3⁺ subpopulation in the subpopulation was measured in FL-5 histogram^; (e) CD4⁺ IL-17A⁺ subpopulation was gated out from lymphocyte by FL-6 and FL-5.

Statistical analysis

Statistical evaluations were performed using JMP^® software ver 11.2.1 (SAS Institute Inc. North Carolina, USA). One-way repeated-measures analysis of variance (ANOVA) with Tukey-Kramer test for multiple comparison, was applied to evaluate the difference between PP-LILI stimuli and placebo stimuli. One-way repeatedmeasures ANOVA with Dunnett test was used to evaluate results from respective days by comparing with the results from day 0 as control. In all tests, a p-value < 0.025 was considered significant and the substantial p-values are stated in figures.

Ethical concerns

Before enrolling the session sequence, we obtained written informed consents from all volunteers. This study was conducted under approval from the ethics review board of Ryotokuji University (approval No. 2304 and 2519).

Results

Expression of IL-10 in lymphocytes became competent to PP-LILI stimulation

To observe the effect of repeated PP-LILI stimuli on IL-10 expression in lymphocytes, 11 volunteers (7 healthy individuals and 4 people with UMC) were enrolled in this study and their IL-10 expression levels in PBMC lymphocytes were observed by flow cytometry. Compared with the IL-10 expression level on the first day (i.e. day 0), significant increases in IL-10 expression were observed at the third weeks of the second and third sessions (day 69 and 112) of PP-LILI stimuli in healthy volunteers. Statistical analysis between Stress-Free therapy (SF) and placebo sessions evidently revealed that those increases of IL-10 expression were consequences of PP-LILI stimuli although the significant induction was not observed in the first session (Figure 2A). In the people with UMC the induction of IL-10 expression was not significantly observed, however, the induction of IL-10 expression in response of PP-LILI stimuli was suggested when the level on day 112 was compared with day 0 (p = 0.031), 91 (p = 0.0100) and 118 (p = 0.0148). When the results obtained from all individuals (n = 11) were analyzed, the results from individuals with UMC did not significantly change the statistical results calculated only from healthy individuals (Figure 2B). The fact that IL-10 expression was not induced in the first sequence of the stimulatory session (i.e. the duration from day 0 to day 56) suggests that induction of PP-LILI-responsive IL-10 expression requires a refractory period that develops competency for the response.

Figure 2.

PP-LILI stimuli induced steep IL-10 expression in lymphocytes of healthy individuals after the second sequence of sessions. Fluorescence intensities from cell-bound anti-IL-10 antibodies were measured and changes of relative mean values over duration were calculated against the mean value obtained on the last day (day 118) because it was lowest. Scattered plots show dynamics of IL-10 expression in lymphocytes over the duration. Comparison of the mean values obtained in consequence of placebo stimuli (including day 0 and 118, shown as open rectangle) and PP-LILI stimuli (shown as filled rectangle) are shown in bar chart. Significant increase in IL-10 expression levels detected by the one-way repeated-measures ANOVA with Dunnett test and Turkey-Kramer test are shown, respectively. (A) IL-10 expression observed in lymphocytes from healthy individuals (solid line) and individuals with UMC (dotted line). (B) IL-10 expression observed in lymphocytes from all volunteers including 7 healthy individuals and 4 people with (n = 11). SF; SF session for PP-LILI stimuli, P; session for placebo stimuli.

Regulatory T cell subsets and proinflammatory IL-17A expressing CD4⁺ T cell subpopulation were not involved in the induction of IL-10 expression in response to PP-LILI stimuli.

Since previous results suggested that PP-LILI stimuli induce distinct IL-10 expression in lymphocytes with the refractory period, we checked the changes that occurred in immunomodulatory lymphocyte subpopulations. Immunosuppressive responses including IL-10 expression are modulated with regulatory lymphocyte subsets such as regulatory T cells (T regs) and regulatory B cells (B regs) ¹¹⁾. T regs are divided into two groups; CD4⁺ T regs ¹²⁾ and CD8⁺ T regs ¹³⁾, and considered to have diverse roles in suppressing autoimmune diseases, and a valance with IL-17A expressing subpopulation of CD4⁺ T cells is important for development of T regs ¹²⁾. So, we tested the populational changes in both CD4⁺ and CD8⁺ T reg cells in addition to IL-17A expressing CD⁴ + T cells. As shown in Figure 3A, CD4⁺ T reg cell subpopulation was randomly changed in their population over the duration and no significant differences related between SF and placebo sessions were observed. CD8⁺ T reg cell subpopulation did not show any significance in changes of their population (Figure 3B). Pro-inflammatory IL-17A expressing CD4⁺ T cell subpopulation seemed to be induced from the second SF session and significantly peaked after the end of PP-LILI stimuli (Figure 3C). These results suggest that T reg cells are not likely to contribute to the development of lymphocytes that are competent for PP-LILI induced IL-10 expression during the refractory period.

Figure 3.

Regulatory T cell subpopulations do not significantly respond on the PP-LILI stimuli. Population changes in both CD4⁺ T regs (A), CD8⁺ T regs (B) and IL-17A expressing CD4⁺ T cells (C) from healthy individuals (solid line) and individuals with UMC (dotted line) were analyzed. No significant differences observed between SF and placebo sessions. (A) Changes in CD4⁺ T reg subpopulation. Scattered plot shows dynamics of the population in CD4⁺ T cells over the duration. Bar chart shows comparison between mean values obtained as results of placebo sessions and SF sessions. (B) Changes in CD8⁺ T reg. Scattered plot shows dynamics of the population in CD8⁺ T cells over the duration. Bar chart shows comparison between mean values obtained as results of placebo sessions and SF sessions. (C) Changes in IL-17A expressing CD4⁺ T subpopulation. Scattered plot shows dynamics of the population in CD4⁺ T cells over the duration. Bar chart shows comparison between mean values obtained as results of placebo sessions and SF sessions. SF; SF session for PP-LILI stimuli, P; session for placebo stimuli.

Activation of CD19⁺ CD24^hi CD38^hi regulatory B cells was distinctly induced by PP-LILI stimulation without refractory period.

Recent studies for immunomodulatory system have shed light to the roles of particular B cells including CD19⁺ CD24^hi CD38^hi and CD19^hi CD1d^hi CD5⁺ B10 cells ^14–17). These cells are named as regulatory B cells (B regs) and the common features of these cells are competence for IL-10 expression ^18–20). B reg cells play indispensable roles to suppress inflammation and autoimmune responses in part through the induction of regulatory T cells and limiting of T helper 1 (Th1) and Th17 cell differentiation ¹⁶⁾. Although previous studies showed the presence and activation of B regs through ex vivo treatment of isolated B cells, it must be detectable in apparently IL-10 expressing lymphocytes though its population is rare. Thus, we tried to detect CD19⁺ CD24^hi CD38^hi cells in PBMC and its activation by observing IL-10 expression. Figure 4A shows that CD24^hi CD38^hi subpopulation (dotted line) was detectable in CD19⁺ cells and was significantly increased on day 56 and 98 that are in the second and third SF sessions in healthy individuals, and the obvious difference between SF and placebo sessions was evaluated. The population of IL-10 expressing CD24^hi CD38^hi subpopulation of CD19⁺ cells, that are activated fraction of B reg cells, was more dynamically changed (solid line in Figure 4A). It was higher in SF sessions and decreased at basal levels during the placebo sessions and the significant differences of its populational change between SF and placebo sessions were detected in both groups of healthy individuals and the people with UMC (Figure 4A and B). Activation of CD19⁺ CD24^hi CD38^hi cells (i.e. increase in relative ratio of IL- 10 expressing cells in the CD¹⁹+ CD24^hi CD38^hi cell population; Figure 4C) confirmed the fact that PP-LILI stimuli induced nearly 100% activation of the population within a week of every SF session (i.e. after second PP-LILI stimulus) for both groups of individuals. Moreover, activation levels of B reg cell subpopulation were obviously down-regulated in the second and third placebo sessions in the group of individuals with UMC, although their background activation levels were rather kept higher until the end of the second SF session (Figure 4C).

Figure 4.

PP-LILI stimuli induce activation of CD19⁺ CD24^hi CD38^hi regulatory B cells rather than increase of its population. Changes in relative population of whole CD19⁺ CD24^hi CD38^hi cells (dotted line and open box) and its IL-10 expressing activated subpopulation (solid line and shaded box) in CD19⁺ B cells in healthy individuals (A) and the people with UMC (B). (C) Changes in IL-10 expressing subpopulation in CD19⁺ CD24^hi CD38^hi regulatory B cells from 7 healthy individuals (solid line and shaded box) and 4 people with UMC (dotted line and open box) were plotted. SF; SF session for PP-LILI stimuli, P; session for placebo stimuli.

Discussion

Our previous study showed that pinpoint plantar longwavelength infrared light irradiation (PP-LILI) stimuli normalizes blood flow and pressure, suppresses ACTH and cortisol levels and improves type II diabetes ^1–3). All these evidences suggested that PP-LILI stimuli affect systemic homeostasis and thus it was thought that the stimuli also affect immune system. Moreover, previous literatures reported that pin-point body surface stimuli such as moxibustion and acupuncture improve inflammatory diseases including ulcerative colitis ^{6, 21)}, pancreatic disorder ⁸⁾ and allergic asthma ^{5, 10)}. Interleukin-10 is produced in several kinds of cells while it is expressed in particular populations of T and B cells in response to inflammation and/or the duration to suppress immune system ^{14, 22)}. Since IL-10 is considered as primary immunomodulator to improve inflammatory diseases, we tested whether PP-LILI stimuli induce IL-10 expression. Although it is preliminary and limited in terms of the scale of clinical trial, our results clearly showed an obvious effect of PP-LILI stimuli to induce IL-10 expression in both healthy individuals and the people with underlying conditions (UMC). Interestingly, this induction of IL-10 expression requires a refractory period during which no response occurred upon the stimuli and thus it suggests a kind of mechanism that leads lymphocytes to be competent for IL-10 expression upon the PP-LILI stimuli. The refractory period seemed to take at least three weeks of SF session followed by non-stimulatory session because of our results. During the refractory period, evident activation of CD19⁺ CD24^hi CD38^hi B reg cells occurred in response to PP-LILI stimuli without long delay (Figure 4C). In addition, activation of CD19⁺ CD24^hi CD38^hi cells was extended in the second and the third Stress-Free Therapy^® (SF) sessions and these activation periods were consistent with the duration of obvious IL-10 expression in major population of lymphocytes. Most probably, these facts together suggest that PP-LILI stimuli primarily activate the B reg cells, and the activated B reg cells consequently potentiate competency of both their progenitor cells and major population of lymphocytes for PP-LILI-responsive IL-10 production.

Meanwhile, the activation of B reg cells and IL-10 expression of lymphocytes steeply decreased to the basal level in the second and third non-stimulatory periods (i.e. placebo sessions) for all individuals regardless of UMC and ages, despite the levels of those activation and expression were rather higher in the people with UMC till the end of the second SF session. The higher levels of B reg cell activation and IL-10 expression in lymphocytes observed in the people with UMC during the refractory phase suggest the counter induction of those subpopulations against the symptoms ^{9, 22, 23)}. Therefore, while the cases of UMC must be checked in larger studies, the evident downregulation of the subpopulations may suggest that the PP-LILI stimuli improve and/or reset the systemic immune responses during the refractory period through the activation of B reg cells.

The competency of CD19⁺ CD24^hi CD38^hi B reg cells were reported to be decreased in elderly people which results in increases of inflammatory disease and autoimmune disorders ^{24, 25)} while we observed evident activations of these cells regardless of ages and some UMCs in response to PP-LILI stimuli (Figure 4C). Here, one must have a primary question how this induction occurred? Because PP-LILI is a mild stimulus that only heat narrow areas of body surface, the linkage between the stimulus on the local tissue such as body surface and the development and activation of B reg cells are missing. Quite recently, Franquesa et al reported that adipose tissue-derived mesenchymal stem cells induce regulatory B cells independently of T helper cells ²⁶⁾. It is conceivable that PP-LILI stimuli activate those mesenchymal stem cells to develop the activated B reg cells through the adipose tissues neighboring stimulatory points. To shed light on the missing linkage, more precise and large-scale study is required.

Our results suggested a possibility that the series of PP-LILI stimuli (i.e. Stress-Free Therapy^®) can serve as not only a potent therapy to treat existing inflammatory diseases but also to prevent the ignition of autoimmune diseases. Meanwhile, the prolonged duration of the B reg cell activation and IL-10 expression in major lymphocytes observed in latter SF sessions might not be welcomed, because, ectopic and/or prolonged suppression of inflammatory responses can deteriorate the prognosis of cancer patients ^27–29). However, obvious down-regulation of activated B reg cells and IL-10 expressing lymphocytes and slight increase of IL-17A expressing CD4⁺ T cell subpopulations after the second SF session (Figure 3C) suggest an ‘balanced’ regulation of systemic immune responses improved by repetitive PP-LILI stimuli. To optimize the systemic response, further analyses for the mechanism and effects induced by PP-LILI stimuli must be required.

In this study, no volunteers were restricted for their daily habits including hot-tub bathing and drinking alcohol. Therefore, the effects we observed in SF sessions should not be ascribed to such habits. Although the whole body heating, similar to the hot-tub bathing, affects our systemic homeostasis and considerable for cardiovascular disorders ^30–33), it is evident that the effect of PP-LILI distinctly predominates over the effects of those daily habits.

Conclusion

In this report, we showed the evidence that PP-LILI stimuli work as a potent inducer to activate CD19⁺ CD24^hi CD38^hi B reg cells and consequently to activate IL-10 expression in major lymphocytes. In addition, along with the repeat of PP-LILI stimuli, activation of B reg cells and IL-10 expression of lymphocytes were obviously down-regulated to the basal level after the termination of the stimuli. The fact that the improvement of higher background of B reg activation and IL-10 expression in lymphocytes in the people with UMC by PP-LILI stimuli is remarkable while the cases of UMC must be checked in larger studies. Since PP-LILI is a non-invasive and moderate treatment without any pain and risks, it must be considerable as an alternative and complementary therapy as curative and analgia for inflammatory diseases. Optimization for treatment parameters and further analyses of the effect and mechanism must be required.

Conflict of interest

Kenji Ryotokuji is an inventor of Stress-Free device^® and holds patents for Stress-Free Therapy^® and interests of Ryoken Co. Ltd that is a manufacturer of the device. Other authors declare that there is no conflict of interests regarding the publication of this paper.